Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development.

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

Deficiency of G protein-coupled receptor Gpr111/Adgrf2 causes enamel hypomineralization in mice by alteration of the expression of kallikrein-related peptidase 4 (Klk4) during pH cycling process.

Enamel is formed by the repetitive secretion of a tooth-specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115-deficient mice show partial enamel hypomineralization, suggesting that other pH-responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111-KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111-deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast-specific protease, kallikrein-related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.

C-terminus of PIEZO1 governs Ca2+ influx and intracellular ERK1/2 signaling pathway in mechanotransduction.

Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca2+ influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca2+ influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs.

Iroquois homeobox 3 regulates odontoblast proliferation and differentiation mediated by Wnt5a expression.

Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1-Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1-Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a, Wnt5a, and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation.

Three-dimensional microscopic comparison of wear behavior between immature and mature enamel: an in vitro study.

BACKGROUND: Dental enamel, the hardest outermost layer of a human tooth, is subjected to occlusal forces throughout life during different oral function as talking, mastication etc. Due to this continuous stress, wear causes the loss of this protective shell. This study aimed to detect microscopic differences in enamel's wear behavior among different age groups of adolescents and adults. AIMS AND METHODS: Enamel specimens from immature open-apex and mature closed-apex premolars were subjected to simulated occlusal wear of impact and sliding wear test ISWT. Upper and lower enamel specimens were made to come in contact under controlled conditions. The enamel specimens' surfaces were examined using different microscopes. The upper and lower specimens were subjected to the following tests; pre-test light microscopy examination, enamel specimens' preparation for ISWT, scanning laser confocal microscopy of upper specimens, three-dimensional (3D) colored laser microscope and a Profilometer imaging of the lower specimens. RESULTS: Wear characteristics, including wear areas, crater depths, and relation to enamel microstructures, differed among different age groups. Immature enamel from the upper specimens was more resistant to chipping than mature enamel with no statistically significant wear area difference. The immature enamel craters from the lower specimens were wider and deeper than those in the mature enamel; the wear areas in the mature enamel in the lower specimens were almost flat and smooth. The wear areas in the immature enamel in the lower specimens were significantly larger than those in the mature enamel. CONCLUSIONS: Wear characteristics of the immature enamel are different from those of the mature enamel. Hence, it should be repaired using restorative materials with compatible wear properties.

von Willebrand factor D and EGF domains regulate ameloblast differentiation and enamel formation.

Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

Transient ventricular bigeminy during emergence from general anesthesia in a pediatric patient with catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare genetic disease that is characterized by ventricular arrhythmias and sudden death, induced by exogenous and endogenous catecholamine. We performed general anesthesia for dental treatment of multiple teeth in a 7-year-old boy with CPVT. To avoid sympathetic tone, anesthesia was maintained by total intravenous anesthesia, but ventricular bigeminy was induced by stimulation on emergence form general anesthesia. Although bigeminy in the present case might have been less likely to induce a fatal arrhythmia, we should keep in mind that a small amount of sympathetic tone may induce arrhythmias in a patient with CPVT.

Cell lineage- and expression-based inference of the roles of forkhead box transcription factor Foxc2 in craniofacial development.

BACKGROUND: Foxc2 is a member of the winged helix/forkhead (Fox) box family of transcription factors. Loss of function of Foxc2 causes craniofacial abnormalities such as cleft palate and deformed cranial base, but its role during craniofacial development remains to be elucidated. RESULTS: The contributions of Foxc2-positive and its descendant cells to the craniofacial structure at E18.5 were examined using a tamoxifen-inducible Cre driver mouse (Foxc2-CreERT2) crossed with the R26R-LacZ reporter mouse. Foxc2 expression at E8.5 is restricted to the cranial mesenchyme, contributing to specific components including the cranial base, sensory capsule, tongue, upper incisor, and middle ear. Expression at E10.5 was still positively regulated in most of those regions. In situ hybridization analysis of Foxc2 and its closely related gene, Foxc1, revealed that expression domains of these genes largely overlap in the cephalic mesenchyme. Meanwhile, the tongue expressed Foxc2 but not Foxc1, and its development was affected by the neural crest-specific deletion of Foxc2 in mice (Wnt1-Cre; Foxc2fl/fl ). CONCLUSIONS: Foxc2 is expressed in cranial mesenchyme that contributes to specific craniofacial tissue components from an early stage, and it seems to be involved in their development in cooperation with Foxc1. Foxc2 also has its own role in tongue development.

G protein-coupled receptor Gpr115 (Adgrf4) is required for enamel mineralization mediated by ameloblasts.

Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.

The transcription factor AmeloD stimulates epithelial cell motility essential for tooth morphology.

The development of ectodermal organs, such as teeth, requires epithelial-mesenchymal interactions. Basic helix-loop-helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD-knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD-KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells, and it may cause inhibition of the cell migration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine whether AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn-KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial-mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development.

The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development.

Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in Nkx2-3, encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that Nkx2-3 is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of Bmp2 and Bmpr2 in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice.

Pannexin 3 inhibits proliferation of osteoprogenitor cells by regulating Wnt and p21 signaling.

Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting ß-catenin degradation through GSK3ß activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca(2+) channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways.

Effect of ethylene oxide unit number in bis-EMA on the physical properties of additive-manufactured occlusal splint material.

PURPOSE: To investigate the effects of the number of ethylene oxide units in bis-EMA on the physical properties of additively manufactured occlusal splints. METHODS: Seven experimental materials containing bis-EMAs with three and 10 ethylene oxide units (BE3 and BE10, respectively) were prepared at different BE10 content rates (BE10-0%, -20%, -30%, -40%, -50%, -60%, and -80%). Half the specimens of each material were aged in boiling water. Flexural strength (FS), flexural modulus (FM), fracture toughness (FT), microwear depth (MD), degree of conversion (DC), water sorption (WSP), water solubility (WSL), color difference between non-aged and aged series (ΔE), and translucency (TP) were evaluated. All the evaluated properties other than FS and MD were analyzed by 1-way ANOVA and Tukey's post hoc analysis, while FS and MD were analyzed by Kruskal-Wallis's test and Bonferroni correction (α=0.05). RESULTS: BE10-80% revealed the lowest FS (P < 0.01 for BE10-0%, -20%, and -30%) and FM (P < 0.01, for all), while revealing the highest DC, WSP, WSL (P < 0.01 for all) and TP (P < 0.01 for all other than BE10-60%). BE10-50% showed the highest FT (P < 0.01 for all). BE10-50%, -60%, and -80% revealed significantly lower ΔE than others (P < 0.01) and lower MD than BE10-0% (P < 0.05). Regardless of the BE10 content, FS, FM, and FT decreased with aging. CONCLUSIONS: The number of ethylene oxide units affects the physical properties of additively manufactured occlusal splints. The higher number of ethylene oxide units in bis-EMA enhanced the microwear resistance, DC, WSP, WSL, color stability, and translucency, whereas it deteriorated the FS and FM.

bmp-2 Gene-Transferred Skeletal Muscles with Needle-Type Electrodes as Efficient and Reliable Biomaterials for Bone Regeneration.

BACKGROUND: Bone morphogenetic protein-2 (bmp-2) has a high potential to induce bone tissue formation in skeletal muscles. We developed a bone induction system in skeletal muscles using the bmp-2 gene through in vivo electroporation. Natural bone tissues with skeletal muscles can be considered potential candidates for biomaterials. However, our previous system using plate-type electrodes did not achieve a 100% success rate in inducing bone tissues in skeletal muscles. In this study, we aimed to enhance the efficiency of bone tissue formation in skeletal muscles by using a non-viral bmp-2 gene expression plasmid vector (pCAGGS-bmp-2) and needle-type electrodes. METHODS: We injected the bmp-2 gene with pCAGGS-bmp-2 into the skeletal muscles of rats' legs and immediately placed needle-type electrodes there. Skeletal tissues were then observed on the 21st day after gene transfer using soft X-ray and histological analyses. RESULTS: The use of needle-type electrodes resulted in a 100% success rate in inducing bone tissues in skeletal muscles. In contrast, the plate-type electrodes only exhibited a 33% success rate. Thus, needle-type electrodes can be more efficient and reliable for transferring the bmp-2 gene to skeletal muscles, making them potential biomaterials for repairing bone defects.

Role of epithelial-stem cell interactions during dental cell differentiation.

Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.

Effect of Surface Polishing on Physical Properties of an Occlusal Splint Material for Additive Manufacturing under Protection Gas Post-Curing Condition.

The aim of this study was to evaluate the effect of surface polishing as well as the post-curing atmospheres (air and nitrogen gas) on the physical properties of an occlusal splint material for additive manufacturing. Flexural strength, flexural modulus, Vickers hardness number (VHN), degree of carbon double bond conversion (DC), water sorption (WSP), and water solubility (WSL) were evaluated. Surface polishing significantly affected the evaluated properties. Regardless of the post-curing atmosphere, flexural strength, flexural modulus, VHN, and DC showed significantly higher values for the polished specimens when compared with the unpolished ones, while WSP and WSL were significantly lower for the polished specimens. Unpolished specimens post-cured at nitrogen gas showed significantly higher VHN and DC values. However, the effect of the post-curing at a nitrogen gas atmosphere was non-significant in polished specimens. The current results suggested that surface polishing plays a role in the physical properties of the evaluated occlusal splint material and can enhance all the evaluated properties regardless of the post-curing atmosphere. Meanwhile, the post-curing at a nitrogen gas atmosphere can enhance the VHN and DC but its effect is confined only to the surface layers, which can be removed during surface polishing.

Wear behavior of materials for additive manufacturing after simulated occlusion of deciduous dentition.

OBJECTIVES: To evaluate wear characteristics of materials for additive manufacturing (AM) after a simulated occlusal test in primary teeth. Wear was simulated by means of impacting - sliding wear testing (ISWT) between specimens prepared from materials for AM against enamel derived from deciduous teeth. METHODS: The prepared hemispherical upper specimens were subjected to impacting-sliding wear test (ISWT) machine against the flattened enamel of deciduous molars on lower specimens. The samples were subjected to 20,000 load cycles using a contact force of 30 N between the opposing surfaces under controlled conditions. In the upper specimens, five groups (n=9): four types of additively manufactured materials Dima, Zenith, Detax, Veltz and a deciduous enamel groups were tested in this study. The enamel-to-enamel group was used as the control. Wear characteristics comprised wear surface area, wear depth, wear volumetric loss, and surface roughness were measured with a confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Data obtained were statistically analyzed by Kruskal-Wallis test and Dunn's test with Bonferroni correction (p < 0.05). RESULTS: Dima showed significantly higher worn surface area (p = 0.009, 0.001, and < 0.001 for Zenith, Detax, and control enamel, respectively), volumetric loss (p = 0.027, 0.007, and < 0.001 for Zenith, Detax, and control enamel, respectively), and damaged opposing enamel (p = 0.002, 0.001, and 0.01 for Detax, Veltz, and control enamel, respectively). There was no significant difference among the volumetric loss in Zenith and Detax. However, SEM revealed that Zenith showed rough worn surfaces and chipping, Detax showed rather a smooth circular worn surface. The worn area of Veltz was smaller than Detax and Zenith at 5,000 cycles, but higher at 15,000 and 20,000 cycles, and SEM showed detachment. CONCLUSION: Wear behavior was different among different materials for AM. In the upper specimens, DM and VZ showed large wear. In the lower specimens, DM caused largest enamel wear and damage. In contrast, ZT and DX showed lower wear and caused less damage to the antagonistic primary enamel. SEM image of ZT showed large losses due to chipping, whereas DX showed the rather smooth. DX was confirmed to have lowest wear and caused least damage to the opposing deciduous enamel, which might be applicable as restorative treatments in deciduous dentition. SIGNIFICANCE: Additive manufactured dental materials could be considered as a treatment modality in deciduous teeth.

Effect of splinting material type and location on resistance against deflection force of splinted periodontally compromised teeth with hypermobility.

This study aimed to evaluate the effect of splinting material type and material location on the force resistance of splinted periodontally compromised teeth with hypermobility. Extracted teeth including the target tooth (maxillary second premolar) and its adjacent teeth were placed into the alveolar sockets of a dental arch model via artificial periodontal ligaments made of elastic impression material. Three different experimental models with varied target tooth mobility including Periotest® values (PTVs) of 20, 30, and 40 were fabricated (named models #20, #30, and #40, respectively). For each experimental model, the force resistance of tooth splinting was tested using the following four materials: everStick PERIO (glass fiber reinforcement: GFR), FORESTAFLEX (braided stainless steel: BSS), Ortho-FlexTech (stainless steel chain: SSC), and Super-Bond (MMA-based resin cement: MRC). The evaluated measures were the PTV after tooth splinting and the required load to cause tooth displacements of 0.05 mm and 0.10 mm in the vertical and lateral directions, respectively. The splinting material type and material location as well as the original PTV of target the tooth significantly affected all the evaluated measures (p < 0.001). MRC revealed the significantly highest force resistance of tooth splinting regardless of material location in each experimental model and was followed by GFR. The PTVs of splinted teeth were comparable to those of adjacent anchor teeth in models #20 and #30 when using GFR, while that was comparable in model #40 when using MRC. Meanwhile, the load causing certain tooth displacement showed a similar tendency to previous-reported data with healthy teeth in model #20 when using GFR, while that showed a similar tendency in models #30 and #40 when using MRC. Overall results concluded that splinting material type and location play a role in the resistance against the deflection force of splinted periodontally compromised hypermobile tooth. It was noted that MRC provided the highest resistance against the deflection force of splinted teeth regardless of material location whereas GFR maintained the physiologically considered tooth mobility.

Development of a novel ex vivo organ culture system to improve preservation methods of regenerative tissues.

Recent advances in regenerative technology have made the regeneration of various organs using pluripotent stem cells possible. However, a simpler screening method for evaluating regenerated organs is required to apply this technology to clinical regenerative medicine in the future. We have developed a simple evaluation method using a mouse tooth germ culture model of organs formed by epithelial-mesenchymal interactions. In this study, we successfully established a simple method that controls tissue development in a temperature-dependent manner using a mouse tooth germ ex vivo culture model. We observed that the development of the cultured tooth germ could be delayed by low-temperature culture and resumed by the subsequent culture at 37 °C. Furthermore, the optimal temperature for the long-term preservation of tooth germ was 25 °C, a subnormothermic temperature that maintains the expression of stem cell markers. We also found that subnormothermic temperature induces the expression of cold shock proteins, such as cold-inducible RNA-binding protein, RNA-binding motif protein 3, and serine and arginine rich splicing factor 5. This study provides a simple screening method to help establish the development of regenerative tissue technology using a tooth organ culture model. Our findings may be potentially useful for making advances in the field of regenerative medicine.

Effect of 3D printing system and post-curing atmosphere on micro- and nano-wear of additive-manufactured occlusal splint materials.

Although additive manufacturing has been widely applied for occlusal splint (OS) fabrication, it is still unclear whether 3D printing system and post-curing atmosphere would play a role in the wear resistance of additive-manufactured OS. Therefore, the aim of this study was to evaluate the effect of 3D printing system (liquid crystal display (LCD) and digital light processing (DLP)) and post-curing atmosphere (air and nitrogen gas (N2)) on the wear resistance of hard and soft OS materials for additive-manufactured OSs (KeySplint® Hard and Soft). The evaluated properties were microwear (by two-body wear test) and nano-wear resistances (by nanoindentation wear test) as well as flexural strength and flexural modulus (by three-point bending test), surface microhardness (by Vickers hardness test), and nanoscale elastic modulus (reduced elastic modulus) and nano surface hardness (by nanoindentation test). For the hard material, the surface microhardness, microwear resistance, reduced elastic modulus, nano surface hardness, and nano-wear resistance were significantly affected by the printing system (p < 0.05), while all the evaluated properties except flexural modulus were significantly affected by the post-curing atmosphere (p < 0.05). Meanwhile, both the printing system and post-curing atmosphere significantly affected all the evaluated properties (p < 0.05). The specimens additive-manufactured by DLP printer tended to show higher wear resistance in the hard material groups and lower wear resistance in the soft material groups when compared to those by LCD printer. The post-curing at N2 atmosphere significantly enhanced the microwear resistance of hard material groups additive-manufactured by the DLP printer (p < 0.05) and soft material groups additive-manufactured by the LCD printer (p < 0.01), while it significantly enhanced the nano-wear resistance of both hard and soft material groups regardless of the printing system (p < 0.01). It can be concluded that 3D printing system and post-curing atmosphere affect the micro- and nano-wear resistance of tested additively manufactured OS materials. In addition, it can be also concluded that the optical printing system providing higher wear resistance depends on the material type, and using nitrogen gas as a protection gas during post-curing enhances the wear resistance of tested materials.